Transcription is the first step in gene expression and the step at which most gene regulation occurs. Transcription in all cells is carried out by multi-subunit RNA polymerases (RNAPs) that are conserved in sequence, structure and function from bacteria to humans. Whereas initiation of DNA synthesis by DNA polymerase requires use of a primer, it is widely accepted that the initiation of RNA synthesis by RNAP occurs de novo (i.e. RNAP initiates RNA synthesis using free NTPs alone). The proposed research will challenge this conventional paradigm. Specifically, we will investigate the hypothesis that a significant fraction of transcription does not occur de novo, but rather relies upon use of small ~2-5 nt RNA transcripts that serve as primers to initiate transcription. We refer to these ~2-5 nt RNA transcripts as nanoRNAs. According to our model, the cell balances nanoRNA-primed transcription and de novo transcription to maintain cellular homeostasis. Furthermore, celular perturbations that alter the balance betwen nanoRNA-primed transcription and de novo transcription lead to global alterations in gene expression. Consistent with this model, we have found that increasing the intracellular concentration of nanoRNAs leads to global alterations in gene expression coupled with an apparent increase in the occurrence of nanoRNA-primed transcription. Building on these findings, we propose to use genetic and biochemical approaches in conjunction with high- throughput sequencing to determine how nanoRNA priming can alter gene expression, identify factors that control the nanoRNA content of the cell, and systematically identify nanoRNAs. The proposed research has the potential to redefine our view of a fundamental process that occurs in all living cells (i.e. transcription) and, in parallel, uncover a novel class of regulatory small RNAs, nanoRNAs, that function in all living cells via a novel mode of action. PUBLIC HEALTH RELEVANCE: The proposed work will investigate the existence of a novel class of regulatory small RNAs, nanoRNAs, that function via a novel mode of action. Small RNAs have emerged as key regulators of cellular homeostasis as well as numerous developmental pathways and disease processes. Thus, the proposed research will lead to discoveries with important implications for public health.